This project is designed to determine the genetic and molecular basis of peptide hormone's cell surface receptor-mediated signal transfer mechanisms. The genetic variations in the regulatory receptor molecules, the nature of hormonal signals, the sequence of events in hormone-induced biological and biochemical responses will be studied by the strategy of mutagenesis and somatic cell genetics in combination with biochemical techniques. Two bioactive polypeptides, insulin and epidermal growth factor (EGF) have been chosen. Insulin and its receptor play important roles in diabetes and some congenital syndromes that mimic aging. EGF is a stimulus for epidermal and epithelial tissue development and a potent mitogen. These hormones are involved in the control of cellular proliferation and have served as basic model systems for the study of physiology and dynamics of ligand/receptor interaction, membrane modulation and malignant transformation. A series of insulin (or EGF)-nonresponsive clones will be selected from in vitro mutagenized cell populations of established cell lines, e.g., 3T3-L1 preadipocyte. A new selection procedure will be applied which utilizes the cytotoxic hybrid molecules cross-linked by disulfide bond between insulin (or EGF) and diphtheria toxin fragment A. In combination with other selection agents, it is expected that various sites of receptor-deficiency will be found in terms of hormone binding, signal transfer mechanisms and biological response. The mutants of mouse origin that have been isolated, e.g., IN-2 and NR-6, and to be isolated, will be fused with human cells expressing functional receptors or subsequent functions or possessing deficiency determined, such as diabetes. The resulting human x mouse cell hybrids will be analyzed with regard to species origin of the expressed phenotypes and the constitution of human chromosomes and genetic markers. Segregation analysis of these parameters in a series of hybrids will permit mapping of human genes responsible for production and regulation of functional receptors and other components. Furthermore, a delivery system of insulin into receptor-deficient cells, e.g., from diabetes will be exploited by using another chimeric insulin cross-linked to ricin B-subunit through ricin binding tests.